The sex difference in hepatic and serum iron, as well as the changes seen in iron metabolism during pregnancy, are attributed to the effects of ovarian hormones on the uptake, mobilization, and storage of iron. We have determined through histochemical means that a sex difference in brain iron exists in the rat and that the levels of iron in the brain appear to change with changing hormone levels whether these fluctuations are naturally occurring or induced. By spectrophotometric means we measured iron levels in the iron-rich globus pallidus and substantia nigra of male and female rats. The effects of naturally fluctuating hormones (as in estrus and pregnancy) on brain iron levels were also determined with this method. Both histochemical and spectrophotometric analysis indicate that a sex difference in brain iron exists, with females having the greater amount, and that brain iron levels vary during estrus and pregnancy.